Sterilized 5-aminolevulinic acid

ABSTRACT

Colored 5-aminolevulinic acid crystals useful for photodynamic therapy are disclosed. Preferably the colored 5-aminolevulinic acid crystals have the color imparted by irradiation of the crystals, such as gamma radiation. The colored ALA crystals are preferably pharmaceutically pure and sterile and can be contained in a sealed sterile container. Also disclosed is sterile aqueous ALA solution which includes the colored ALA crystals contained in water. Also disclosed is a method for preparing colored ALA crystals which includes exposing non-irradiated ALA crystals to a radiation source at a dose sufficient impart a color which is different than any color present in the non-irradiated crystals. Preferably the irradiation is sufficient to sterilize the ALA crystals. The sterile colored ALA crystals can be used in a kit for internal or external treatment and/or detection of a condition in a mammal, which includes the sterile, colored ALA crystals and sterile diluent, and in the case of internal treatment and/or detection optionally a catheter for administration of the ALA.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates generally to the field of medicine, particularlypharmacotherapeutics and pharmacodetection, using photosensitizingagents and precursors thereof, especially 5-aminolevulinic acid, alsoknown by the acronym "ALA." More specifically, this invention relates tocolored ALA, especially sterilized ALA, which is stable under commercialconditions.

2. Description of Related Art

Photodynamic therapy involves the administration of a photosensitizingagent to a subject, including administration of a precursor of aphotosensitizing agent such as ALA, and subsequent irradiation withlight of the target cells or tissue of the subject. The photosensitizingagent preferentially accumulates in the target cells, namely cells ortissues that are more rapidly proliferating or growing than other cellsor tissues in the target environment. The target cells may be morerapidly proliferating because they are malignant or non-malignant, ofinfective agent origin, e.g. viral, bacteria, parasite or fungal originor not of infective agent origin; are normally hyperproliferative, suchas the endometrium of pre-menopausal women, or are abnormallyhyperproliferative, such as cells infected with an infective agent.

Although not intending to be bound by any particularly theory, it hasbeen proposed that following administration of ALA, as a result of theirmore rapid proliferation, the target cells or tissue contain relativelygreater concentrations of light sensitive porphyrins and thus are moresensitive to light.

The target cells or tissue containing sufficiently high concentrationsof the photosensitizing agent, including the metabolites of ALA,selectively absorb greater amounts of light and can be selectivelylocalized and distinguished from the adjacent cells or tissues viafluorescence, or damaged or destroyed. The effect of the light, as iswell known in the art, depends on the photosensitizer selected; thewavelength, intensity and duration of administration of the light; andthe timing of irradiation vis-a-vis the administration of thephotosensitizing agent, and results in fluorescence or impairment ordestruction of the target cells or tissue.

A variety of photosensitizing agents have been used for photodynamictherapy. The only commercially available photosensitizing agent isPhotofrin™, a hemato porphyrin or HPD, sold by QLT Phototherapeutics,Inc. Vancouver, British Columbia. Synthetic porphyrins often have thedisadvantage of having longer half-lives and lowered sensitivity to therapidly growing cells as contrasted with normal cells than do naturallyoccurring porphyrins. The half-life of the photosensitizing agent issignificant, since the buildup of excess porphyrins in the skin canresult in reddening or burning of the skin.

An alternative to synthetic porphyrins are natural porphyrins. Naturalporphyrins appear to have shorter half-lives than their syntheticcounterparts, but are difficult to synthesize and more importantly, areunstable ex vivo under environmental conditions to which drugs aresubjected in normal commercial distribution and storage channels.

A revolutionary discovery made in the late 1980's was that the naturallyoccurring amino acid ALA, a precursor in the metabolic pathway to heme,could be used in photodynamic therapy instead of synthetic porphyrins.ALA appears to act in the body as a precursor to naturally occurring,light sensitive porphyrins, which avoids the ex vivo problems associatedwith natural porphyrins noted above. This discovery has broughtphotodynamic therapy to a world wide interest level never beforeachieved with synthetic porphyrins.

ALA has a very short half-life, depending on the route ofadministration, is highly tissue specific and, as a naturally occurringamino acid, minimizes complications and side effects which arise whenforeign substances are administered to the body. Unlike syntheticporphyrins, ALA also makes it possible to distinguish small or flattumors, e.g. in the bladder, from normal tissues, visually by means offluorescence excitation.

It is believed that ALA is converted by the cells and tissues in vivo orex vivo into protoporphyrin IX and related endogenous biochemicals,which fluoresce or are degraded by light of the appropriate wavelength.The preferential accumulation of such naturally occurring porphyrins inrapidly growing cells permits the targeting of such cells.

One of the roadblocks to the commercial use of ALA has been its extremeliability to destruction under ambient conditions. Aqueous solutions ofALA maintained under ambient conditions are progressively, degradedquite rapidly, resulting in degradation products, primarily 2,5-pyrazinedipropionic acid and intermediate degradation products which have notbeen able to be identified due to their transient nature. However, theintermediate degradation products are believed to include 2,5(beta-carboxymethyl)-dihydropyrazine. FIG. 1 depicts the degradation ofALA to 2,5 (beta-carboxymethyl)-dihydropyrazine and then to 2,5-pyrazinedipropionic acid. Formulating ALA in nonaqueous creams and gels did notprevent this degradation. Even ALA pressure-sensitive adhesive mixturesdid not totally stop oxidative reactions. Likewise, the preparation ofpharmacological equivalents of ALA such as functional derivatives of thecarboxylic acid group, substitution of the amino group, blocking of theoxo group or the use of simple or more complex acid addition, acid, baseor neutral salts has not completely overcome this problem because themore stable the product, the greater effect there may be on themetabolism of the product in the body.

SUMMARY OF THE INVENTION

One object of the invention is to provide ALA which does not suffer fromthe problems of the known art, particularly the extreme degradation ofALA. Another object of the invention is to provide sterile stable ALAwhich is pharmacologically active and can be used in photodynamictherapy and detection. Still another object of the invention is toprovide a method for making sterile, stable ALA which does not sufferfrom the problems of the known art. Yet another object of the inventionis to provide a combination of ALA and an endoscope for internal use ina mammal. Still another object of the invention is to provide a methodfor using ALA in the detection and/or treatment of a condition in amammal.

The foregoing objects and other objects are achieved according to oneaspect of the present invention by colored 5-aminolevulinic acid,preferably 5-aminolevulinic acid HCl. In a preferred embodiment, thecolor is imparted by irradiation of crystals, preferably gammaradiation. According to another aspect of the invention, there has beenprovided colored 5-aminolevulinic acid crystals having an F-center pointdefect in the crystal lattice, where said F-center point defect impartssaid color to the crystals.

According to still another aspect of the invention, there has beenprovided a sterile aqueous ALA solution comprising the colored ALAcrystals according to the invention, contained in water. According toyet another aspect of the invention, there has been provided a sterilepackage comprising colored ALA crystals according to the invention in asealed sterile container. According to yet another aspect of theinvention, there has been provided a method for preparing colored ALAcrystals according to the invention, which includes exposingnon-irradiated ALA crystals to a radiation source at a dose sufficientto impart a color which is different than any color present in thenon-irradiated crystals. According to still another aspect of theinvention, there has been provided a kit for internal and/or externaltreatment and/or detection of a condition in a mammal, which includesthe sterile colored ALA crystals according to the invention and asterile diluent, preferably water. In one mode of internal treatmentand/or detection, the kit optionally includes a catheter and optionallyan endoscope.

According to still another aspect of the invention, there has beenprovided a method of administering 5-aminolevulinic acid in a stableform for internal and/or external mammal administration which comprisesthe administration of a solution of ALA derived from the colored ALAaccording to invention.

Further objects, features and advantages of the present invention willbecome apparent from consideration of the detailed preferred embodimentswhich follow.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1-12 compare gamma irradiated with nonirradiated crystalline ALA.In the figures, "REF" denotes nonirradiated ALA and "IRR" denotesirradiated ALA.

FIG. 1 depicts the degradation reaction pathway of ALA.

FIG. 2 compares thermogravimetic measurements.

FIG. 3. compares differential scanning calorimetry measurements.

FIG. 4 compares reflectance spectra of solid samples.

FIG. 5 compares the photoacoustic spectroscopy ("PAS") spectra.

FIG. 6 compares the PAS spectra of gamma irradiated ALA as a function ofthe irradiation dose.

FIG. 7 compares the PAS spectra of a single sample of ALA before and 20minutes after irradiation with visible light at 410 nm (2 mW cm⁻²) andafter a dark period of 10 days.

FIG. 8 compares the reflectance spectra before and 2.2 hours afterirradiation with visible light at 410 nm (0.12 mW cm⁻²) and after a 2day dark period.

FIG. 9 compares the reflectance spectra before and after dissolution inmethanol and evaporation of the solvent of ALA irradiated at 25 kGy.

FIG. 10 compares the reflectance spectra prior to and after dissolutionin methanol and evaporation of the solvent for a different set ofsamples than used in FIG. 9 irradiated at 25 kGy.

FIG. 11 compares the absorbance spectra of both gamma-irradiated andreference ALA in methanol for ALA which was stored at -10° C. and at 40°C.

FIG. 12 compares the absorbance spectra of both gamma-irradiated andreference ALA upon the first, and second dissolution in 0.2M PBS, pH 5for ALA stored at both -10° C. and 40° C.

FIG. 13 shows a photograph taken at 4× magnification of gamma-irradiatedcolored ALA crystals.

FIG. 14 shows a photograph taken at 4× magnification of non-irradiatedALA crystals.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

5-Aminolevulinic acid is also known as 5-aminolaevulinic acid,δ-aminolevulinic acid, δ-aminolaevulinic acid and 5-amino-4-oxopentanoicacid. 5-Aminolevulinic acid can be used as the salt, particularly asimple salt and especially the hydrochloride salt. 5-Aminolevulinic acidcan also be used in the form of a precursor or product of5-aminolevulinic acid. 5-Aminolevulinic acid can also be used in itspharmacologically equivalent form, such as an amide or ester. Examplesof precursors and products of 5-aminolevulinic acid andpharmacologically equivalent forms of 5-aminolevulinic acid that can beused in the present invention are described in J. Kloek et al., Prodrugsof 5-Aminolevulinic Acid for Photodynamic Therapy, Photochemistry andPhotobiology, Vol. 64 No. 6, December 1996, pages 994-1000; WO 95/07077;Q. Peng et al., Build-Up of EsterifiedAminolevulinic-Acid-Derivative-Induced Porphyrin Fluorescence in NormalMouse Skin, Journal of Photochemistry and Photobiology B: Biology, Vol.34, No. 1, June 1996; and WO 94/06424, which are all incorporated byreference herein in their entirety. As used herein, all of thesecompounds, unless other wise noted, are referred to jointly andseverally as "ALA."

As used herein, the term "sterilized" refers to a product which has beenprocessed to make it suitable for internal pharmaceutical use.

As used herein, the term "colored" is defined as color that has beeninduced by irradiation. This is to be distinguished from non-irradiatedALA which is generally white, but in some instances may be off-white,probably due to the presence of varying amounts of degradation products.

As used herein, a "pharmaceutically pure" substance is defined as asubstance which is suitable for therapeutic and detection use in humansand other mammals. Preferably, the pharmaceutically pure substance islabeled for therapeutic and detection use in humans and other mammals.

The present invention is based on the finding that sterile ALA can bemade and prepared well in advance of its final use, despite the extremedegradation generally found with ALA as noted above. It was further andsurprisingly found that despite the yellow color of irradiatedcrystalline ALA, the substance had not been degraded, despite thedamaging effects that radiation, particularly gamma radiation, can haveon thermodynamically unstable substances. In addition, it has beensurprisingly found that the gamma irradiated material is stable forextended periods of time, at least one year, when stored in the sealedbottle in which it was irradiated.

The sterilized colored ALA crystals are preferably substantially free ofimpurities, such as degradation products and pyrogens, especially whenintended for systemic administration. The amount of impurities isgenerally ≦2.0 wt %, preferably ≦1.0 wt %, and more preferably ≦0.5 wt%.

The crystalline ALA is colored to the naked eye by irradiation from asource which emits electromagnetic radiation, preferably gamma radiationor other ionizing radiation. For example when the hydrochloride salt ofALA is employed, the irradiation alters the appearance of the crystalsto the naked eye from white or off-white to yellow. The intensity of thecolor, such as the yellow color for ALA HCl, is dependent on thecrystalline form that is irradiated, such as the form as commerciallysupplied or a crystal that is further micronized. For example, when theALA crystals are micronized, the irradiated ALA micronized crystals areless intensely colored than intact (i.e., non-micronized) ALA crystals.This is consistent with the F-center theory for colorization describedbelow. The intensity is also dependent on the dosage of radiation thatthe crystalline ALA has been supplied with.

This color disappears after dissolution of the irradiated crystals anddoes not appear again upon recrystallization from an aqueous solutionunder ambient (i.e., room temperature and pressure) conditions. Thecolored crystalline irradiated ALA has not been distinguished in anychemical property from non-irradiated ALA, either by spectroscopic,chromatographic, solution pH or solubility profile, except for the colorof the irradiated material. This is explained more fully below withrespect to FIGS. 2 to 14.

Gamma irradiated ALA, as can be seen from FIGS. 2 through 14 issubstantially physically and chemically the same, except for the color,as the non-irradiated ALA with respect to spectroscopic,chromatographic, solution pH and solubility profile. Although notintending to be bound by any theory, it appears that the color impartedby irradiation is due to F-centers.

The calorimetric measurements of FIGS. 2 and 3 indicate that any majorstructural differences are present in quantities of less than 1% of thetotal ALA amount, or that the type of any structural modification issuch that the sensitivity of the calorimetric assay to the change isbelow the capacity of the instrumentation.

Spectral reflectance as shown in FIGS. 4 and 8-10 is very sensitive tospectral differences at or near the surface, while photoacousticspectroscopy ("PAS") as shown in FIGS. 5-7 is sensitive to changes bothat the surface and the interior of the crystal. Both of these techniquesrevealed significant spectral differences between the irradiated andnonirradiated reference samples of ALA in the solid crystalline phase.Since no differences were observed in the solution phase spectra, theyellow color of the irradiated material is likely to be a property ofthe crystalline solid only. Calorimetry failed to show gross crystalmodifications, supporting the view that the crystal modifications aresuch that the overall crystal symmetry is not significantly affected.

Although not wishing to be bound by any theory, gamma radiation is knownto cause point defects in substances such as alkali halides. Suchalterations do not change the overall symmetry of the crystal, sincethey involve only the removal or relocation of small numbers of specificions, while essentially leaving the major crystalline structure intact.The most common type of point defect caused by the ionizing radiation ofthe F-(farben)-center, which is a negative ion vacancy with one excesselectron bound at the vacancy. The time required to fill the vacancyformed by the electrons is on the order of minutes to days, and isdependent on the diffusion rate of electrons in the crystal lattice.F-centers have been extensively studied and are characterized by one ormore absorbance band(s) at higher wavelengths (lower energy) than thatof the normal absorbance of the molecules surrounding the excesselectron. These allowed, red-shifted bands are believed to stem from theexcess electron gaining state function properties from the surroundingmolecules in a symmetrical manner. The location of the UV-visibleabsorbance bands seen in FIGS. 4, 5 and 6, detected by both PAS andreflectance spectroscopy, is consistent with the F-centers.

The spectra shown in the photobleaching studies of FIGS. 7 and 8 isconsistent with this F-center theory. The spectra depicted in FIGS. 9through 12, suggest that the color is due to an F-center defect ratherthan preformed chemical degradation. FIGS. 13 and 14 indicate that nomicroscopic changes take place upon irradiation of the ALA.

Moreover, when ALA is irradiated, there is generally some delay beforecoloring of the crystal sets in. As noted above, the coloring is due toan ion being "kicked out" during irradiation, leaving a charged hole.The temporal delay in coloring is believed due to the basis of ionmigration through the crystal lattice. This delay further supports theF-center theory.

Although the above measurements and analysis were carried out on ALAHCl, which produces a yellow color, it is fully expected that othercrystalline forms of ALA would also experience non-degradation relatedcoloring upon irradiation.

Another aspect of the invention provides methods for the preparation ofsterile ALA suitable for internal use in human subjects and othermammals, namely by irradiating, particularly γ-irradiation, of ALA.Preferably, the irradiation is carried out in a sealed container, suchthat both the ALA and the container are sterilized during irradiation.The irradiation procedure sterilizes the ALA in the conventional sense.Sterilization by radiation, particularly gamma radiation is well knownin the art and will not be discussed at length. More detailedinformation can be found in Gamma Processing Technology: An AlternativeTechnology for Terminal Sterilization of Parenterals, by B. D. Reid, PDAJournal of Pharmaceutical Science & Technology, vol. 49, no. 2,March-April 1995, pages 83-89, which is incorporated herein in itsentirety.

The ALA is irradiated with a dose of radiation sufficient forsterilization. A sufficient dose of radiation can be determined bymethods known to those skilled in the art. For example, afterirradiating ALA with a selected dose, the ALA can be transferred to theappropriate media for encouraging the growth of viable microorganisms todetermine if sterilization is sufficiently complete.

For most applications using gamma irradiation, a dose of 5 kilograys orgreater has been found to provide sufficient sterilization. However, asnoted above, the crystalline ALA has been surprisingly found to resistdegradation even at high doses of gamma radiation. Thus, it is possibleto provide a dose of 25 kilograys or greater, without detectabledegradation of the ALA. This is significant, in that the United StatesFood and Drug Administration's ("FDA") level of gamma irradiation for"overkill" is 25 kilograys. This is a level at which the FDA presumes,without evidence, that virtually all microorganisms have been killed.

The present invention also provides for methods of using the sterilizedALA in photodynamic therapy internally or externally on the tissues orcells of a mammal. For external use, the sterilized ALA may be appliedby an applicator. For internal use, application may be orally,intravenously or administration by a catheter. For example, theadministration of the ALA can be on internal surfaces of the body of thesubject, typically in conjunction with an endoscope coupled to a lightsource. Numerous publications discuss photodynamic therapy, see e.g.Kriegmair et al., "Photodynamic Diagnosis (PDD) for Early Recognition ofCarcinomata of the Bladder", ENDO World Uro No. 17-E, 1955, apublication of Karl Storz Gmbh & Co. and the equipment referred totherein. See also:

1. Bahnson R. R. Editorial: Urothelial malignancy - Much promise butlittle progress. J. Urology 1996; 155:122.

2. Baumgartner R., Kriegmair M., Jocham D., Hofstetter A., Huber R,,Karg O., Haussinger K. Photodynamic diagnosis (PDD). of early stagemalignancies - Preliminary results in urology and pneumology. SPIE 1992;1641:107-112.

3. Baumgartner R., Kriegmair M., Lumper W., Riesenberg R., Stocker S.,Sassy T., Hofstetter A. ALA-assisted fluorescence detection of cancer inthe urinary bladder. SPIE 1993:2081 (International Symposium onBiomedical Optics, September 1993, Budapest, Hungary)

4. Chang S-C, MacRobert A. J., Bown S. G. The biodistribution andphotodynamic effect of protoporphyrin IX in rat urinary bladders afterintravesical instillation of 5-aminolaevulinic acid. SPIE 1995;2371:289296.

5. Chang S-C, MacRobert A. J., Bown S. G. Biodistribution ofprotoporphyrin IX in rat urinary bladder after intravesical instillationof 5-aminolaevulinic acid. J. Urology 1996;155:1744-1748.

6. Chang S-C, MacRobert A. J., Bown S. G. Photodynamic therapy on raturinary bladder with intravesical instillation of 5-aminolaevulinicacid: light diffusion and histological changes. J. Urology 1996;155:1749-1753.

7. Forrer M., Glanzmann T., Mizeret J., et al. Fluorescence excitiationand emission spectra of ALA induced protoporphyrin IX in normal andtumoral tissue of the human bladder. SPIE 1995; 2324:84-88.

8. Iinuma S., Farshi, S. S., Ortel B., Hasan T. A mechanistic study ofcellular photodestruction with 5-aminolevulinic acid-induced porphyrin.Br. J. Cancer 1994; 70:21-28.

9. Iinuma S., Bachor R., Flotte T., Hasan T. Biodistribution andphotoxicitiy of 5-aminolevulinic acid induced PpIX in an orthotopic ratbladder tumor model. J. Urology 1995; 1 53:802-806.

10. Jichlinski P. P., Mizeret J., Forrer M., Wagniere G., Van den BerghH., Schmidlin F., Graber P., Leisinger H-J. Les tumeurs superficiellesde la vessie. Rappel pathologique et clinique, et presentation d'unenouvelle methode diagnostique: la photodetection par fluorescence descarcinomas a epithelium de transition basee sur I'induction deprotoporphyrine IX par I'acide delta-aminolevulinique (5-ALA).

11. Jichlinski P., Forrer M., Mizeret J., Braichotte D., Wagnieres G.,Zimmer G., Guillou L., Schmidlin F,, Graber P., Van den Bergh H.,Leisinger H-J. Usefulness of fluorescence photodetection of neoplasticurothelial foci in bladder cancer following intravesical instillation ofdelta-aminolevulinic acid (5-ALA). SPIE 1996; 2671:340-347.

12. Jichlinski P., Forrer M., Mizeret J., Glanzmann T., Ddraichofte D.,Wagnieres G., Zimmer G., Guillou L., Schmidlin F., Graber P., Van denBergh H., Leisinger H-J. Clinical evaluation of a method for detectingsuperficial transitional cell carcinoma of the bladder by light inducedfluorescence of protoporphyrin IX following topical application of5-aminolevulinic acid. Preliminary results. Lasers in Surgery andMedicine 1996;ln review.

13. Jocham D., Baumgartner R., Fuchs N., Lenz H., Stepp H., Unsold E.Die fluoreszenzdiagnose porphyrin-markierter utothelialer tumoren.Urologe (A) 1989; 28:59-64.

14. Jocham D. Photodynamische verfahren in der urologie. Urologe 1994;3:547-552.

15. Kriegmair M., Baumgartner R., Hofstetter A. Intravesikaleinstillation von delta-aminolavulinsaure (ALA) - eine neue methode zurphotodynamischen diagnostik und therapies Lasermedizin 1992; 8:83.

16. Kriegmair M., Baumgartner R., Knuchel R., Ehsan A., Steinbach P.,Lumper W., Hofstadter F., Hofstetter A. Photodynamische diagnoseurothelialer neoplasien nach intravesikaler instillation von5-aminolavulinsaure. Urologe 1994; 33:270-275.

17. Kriegmair M., Baumgartner R.,, Knuechel R., Steinbach P., Ehsan A.,Lumper W., Hofstadter F., Hofstetter A. Fluorescence photodetection ofneoplastic urothelial lesions following intravesical instillation of5-aminolevulinic acid. Urology 1994; 44:836-841.

18. Kriegmair M., Baumgartner R., Ehsan A., Lumper W., Hofstetter A.,Knuechel R., Steinbach P., Hofstadter F. Detection of early bladdercancer and dysplasia by fluorescence cystoscopy. J. Urology 1995;153:457 A.

19. Kriegmair M., Stepp H., Steinbach P., Lumper W., Ehsan A., Stepp H.G., Rick K., Knuchel R., Baumgartner R., Hofstetter A. Fluorescencecystoscopy following intravesical instillation of 5-aminolevulinic acid:a new procedure with high sensitivity for detection of hardly visibleurothelial neoplasias. Urol lnt 1995;55: 190-196.

20. Kriegmair M., Baumgartner R., Knuchel R., Stepp H., Hofstadter F.,Hofstadter A. Detection of early bladder cancer by 5-aminolevulinic acidinduced porphyrin fluorescence. J. Urology 1996; 155:105-110.

21. Kriegmair M., Baumgartner R., Lumper W., Waidelich R., Hofstetter A.Early clinical experience with 5-aminolevulinic acid for photodynamictherapy of superficial bladder cancer. British Journal Urology 1996;Accepted for publication.

22. Kriegmair M., Baumgartner R., Lumper W., Riesenberg R., Stocker S.,Hofstetter A. Fluorescence cystoscopy following intravesicalinstillation of aminolevulinic acid (ALA). 204 A.

23. Kriegmair M., Baumgartner R., Susanne S., Riesenberg R., HofstetterA., Knuchel, R., Steinbach P. Photodynamic treatment of urothelialcancer following intravesical application of 5-aminolaevulinic acid in arat bladder tumor model. J. Urology 1994, 151: 518A, abstract 1163.

24. Kriegmair M., Lumper W., Hofstetter A., Stenzl A., Holtl L., BartschG. Photodynamic therapy of superficial bladder cancer based onintravesical application of 5-aminolevulinic acid. Proceedings of theAmerican Urological Association 1996; 155: 566A.

25. Kriegmair M., Stepp H., Baumgartner R., Hofstetter A., Knuchel R.,Steinbach P., Hofstadter F. Fluorescence controlled transurethralresection of bladder cancer following intravesical application of5-aminolevulinic acid. Proceedings of the American UrologicalAssociation 1996;155: 655A.

26. Leveckis J., Burn J. L., Brown N. J., Reed M. W. R. Kinetics ofendogenous protoporphyrin IX induction by aminolevulinic acid:preliminary studies in the bladder. J. Urology 1994; 152:550-553.

27. Moore R. B., Miller G. G., Brown K., Bhatnagar R., Tulip J., McPheeM. S. Urothelial conversion of 5-aminolevulinic acid toprotoporphyrin-IX following oral or intravesical administration. SPIE1995; 2371:284-288.

28. Novo M., Huttmann G., Diddens H. Chemical instability of5-aminolevulinic acid used in the fluorescence diagnosis of bladdertumours. J. Photochem. Photobiol. B: Biol. 1996; Accepted forpublication.

29. Rodriguez M., Huttmann G., Diddens H. Chemical instability of 5aminolevulinic acid (ALA) in aqueous solution. SPIE 1995; 2371:204-209.

30. Thomas S., Kaspers I., Schmitt-Conrad M., Svanberg K., Diddens H.,Huttman G., Jocham D. Photodynamic imaging of urothelial bladder cancerafter topical instillation of 5-aminolevulinic acid (5-ALA), 5thBiennial Meeting of the International Photodynamic Association,September 1994, Amelia Island, Fla. (U.S.A.).

31. Steinbach P., Kriegmair M., Baumgartner R. Hofstadter F. Z. KnuchelR. Intravesical instillation of 5-aminolevulinic acid: the fluorescentmetabolite is limited to urothelial cells. Urology 1994: 44:676-681

32. Steinbach P., Weingandt H., Baumgartner R., Kriegmair M., HofstadterF., Knuichel R. Cellular fluorescence of the endogenous photosensitizerprotoporphyrin IX following exposure to 5-aminolevulinic acid.Photochem. Photobiol. 1995;62:887-895.

All of the above references are incorporated herein in their entireties.Other photodynamic therapy or photodetection uses include treatment ofactinic keratoses, hair removal, treatment of acne and endometrialablation.

Endoscopes coupled to a light source for use in photodynamic therapy aresold commercially but can be especially designed for use with ALA andits precursors. Suitable endoscopes for use with a light source arecommercially available, e.g. from Karl Storz Gmbh & Co., Tuttligen,Germany; Circon ACMI; Olympus; and Richard Wolf. Another suitableendoscope is described in U.S. Pat. No. 5,441,531, incorporated hereinby reference in its entirety.

This invention also provides for commercial kits containing sterilizedALA, a sterile diluent such as an aqueous buffer solution, and,optionally a catheter for administering the ALA solution. An endoscopecoupled to a light source such as those described above, for use indetection or treatment of the cells or tissues in which the ALApreferentially accumulates can also be included in the kit. Preferablyinstructions on the use of the ALA are packaged with the kits.

Reference will now be made to the following non-limiting examples.

EXAMPLES 1-4 and COMPARATIVE EX. 1

ALA hydrochloride salt was obtained from Sochinaz, S. A., Vionnez,Switzerland. 1.65 grams of ALA hydrochloride salt was placed into 60 mlglass vials. 360 vials of crystalline non-irradiated ALA were prepared.Of these, 225 vials were sealed under ambient conditions, and wereirradiated with gamma radiation as shown in Table 1.

                  TABLE 1    ______________________________________                                  target dose    Example no. of vials                      conditions  (KGys) color    ______________________________________    1       45        crystalline,                                  15     yellow                      atmospheric,                      room    2       45        same as Ex. 1                                  30     yellow    3       45        crystalline,                                  15     yellow                      atmospheric,                      packed in dry ice    4       45        same as Ex. 3                                  30     yellow    Comp. Ex. 1            45        same as Ex. 1                                  0      off white    ______________________________________

EXAMPLES 5-6 AND COMPARATIVE EX. 2

The other 135 vials as prepared above were sealed under an argonatmosphere and were irradiated as shown in Table 2.

                  TABLE 2    ______________________________________                                  target dose    Example no. of vials                      conditions  (KGys) color    ______________________________________    5       45        crystalline, argon                                  15     yellow                      atmosphere,                      room temperature    6       45        same as Ex. 5                                  30     yellow    Comp. Ex. 2            45        same as Ex. 5                                  0      off white    ______________________________________

EXAMPLES 7-10 AND COMPARATIVE EXS. 3 and 4

Crystalline ALA was first micronized by pulverizing into fine particlesa few μm in diameter by a jet mill by Micro-Macinazione, S.A. todetermine if further reducing the crystalline size would have any effecton the color of the irradiated ALA. After micronization, 1.65 grams ofALA hydrochloride salt was placed into 60 ml glass vials. A total of 270vials of crystalline non-irradiated ALA were prepared. Of these, 135vials were sealed under ambient conditions and irradiated with gammaradiation as shown in Table 3. The other 135 vials were sealed under anargon atmosphere and irradiated with gamma radiation as shown in Table3.

                  TABLE 3    ______________________________________                                  target dose    Example no. of vials                      conditions  (KGys) color    ______________________________________    7       45        micronized  15     yellow                      crystalline,                      atmospheric,                      room temperature    8       45        micronized  30     yellow                      crystalline,                      atmospheric,                      room temperature    Comp. Ex. 3            45        micronized  0      off white                      crystalline,                      atmospheric,                      room temperature    9       45        micronized  15     yellow                      crystalline,                      argon, room                      temperature    10                micronized  30     yellow                      crystalline,                      argon, room                      temperature    Comp. Ex. 4       micronized  0      off white                      crystalline,                      argon, room                      temperature    ______________________________________

Subsequent analysis of the type described above with respect to FIGS.2-14, confirmed that there was no discernible difference in structure orpharmacological activity between the irradiated examples and thenon-irradiated control comparative examples.

The gamma irradiation also results in a darkening of the glass bottleused to contain the ALA. Presumably the darkening is due to impuritiesin the glass that do not affect its pharmaceutical acceptability. Infact, this darkening is an advantage since it shades the bottle contentsfrom direct light.

Other embodiments of the invention will be apparent to those skilled inthe art from consideration of the specification and practice of theinvention disclosed herein. It is intended that the specification beconsidered as exemplary only, with the true scope and spirit of theinvention being indicated by the following claims.

What is claimed is:
 1. Colored 5-aminolevulinic acid crystals, whereinthe color is imparted by irradiation of the crystals.
 2. The colored ALAcrystals according to claim 1, wherein said crystals arepharmaceutically pure and sterile.
 3. The colored ALA crystals accordingto claim 1, wherein said irradiation is gamma irradiation.
 4. Thecolored ALA crystals according to claim 3, wherein said gammairradiation is sufficient to sterilize said crystals.
 5. The colored ALAcrystals according to claim 4, wherein said gamma irradiation provides adose of at least 5 kilograys.
 6. The colored ALA crystals according toclaim 4, wherein said gamma irradiation provides a dose of at least 25kilograys.
 7. The colored ALA crystals according to claim 1, whereinsaid ALA contains less than 2.0 wt % impurities.
 8. The colored ALAcrystals according to claim 1, wherein said ALA contains less than 1.0wt % impurities.
 9. The colored ALA crystals according to claim 1,wherein said ALA contains less than 0.5 wt % impurities.
 10. The coloredALA crystals according to claim 1, wherein said crystals dissolved in anaqueous solution have substantially the same spectroscopic,chromatographic, solution pH and solubility profile as nonirradiated5-aminolevulinic acid.
 11. A sterile ALA solution comprising the coloredALA crystals according to claim 2, contained in a diluent.
 12. A sterilepackage comprising colored ALA crystals according to claim 2 in a sealedsterile container.
 13. A sterile package according to claim 12, whereinthe container is sterilized by irradiation.
 14. A method for preparingcolored ALA crystals according to claim 1, comprising exposingnon-irradiated ALA crystals to a radiation source at a dose sufficientto impart a color which is different than any color present in thenon-irradiated crystals.
 15. A method for preparing colored ALA crystalsaccording to claim 14, further comprising: placing non-irradiated ALAcrystals into a container; sealing said container; and exposing thenon-irradiated ALA crystals and the container to a radiation source at adose sufficient to impart a color which is different than any colorpresent in the non-irradiated crystals and is sufficient to sterilizesaid crystals and container.
 16. A kit for internal treatment and/ordetection of a condition in a mammal, comprising the sterile colored ALAcrystals according to claim 2 and sterile diluent.
 17. A kit accordingto claim 16, further comprising a catheter.
 18. A kit according to claim16, wherein the colored ALA crystals are packaged in a sterilecontainer.
 19. A method of administering 5-aminolevulinic acid in astable form for internal and/or external mammal administration whichcomprises the administration of a solution of ALA derived from thecolored ALA according to claim
 1. 20. Colored 5-aminolevulinic acidcrystals comprising an F-center point defect in the crystal lattice,wherein said F-center point defect imparts said color to the crystals.